The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Prostate-specific antigen (PSA) is an abundant 33 kDa glycoprotein secreted by the epithelial cells of the prostate (Brawer, Acta Oncol 1991; 30:161-8). The concentration of PSA in serum is normally very low but known to frequently increase above normal (i.e. 4 .mu.g/L) in prostate cancer (CAP) patients. Therefore, immunoassys measuring the serum PSA are routinely used to monitor disease progress and relapse of CAP after removal of prostate (Brawer et al. Urology 1989; 5(suppl):11-6, Oesterling J Urol 1991; 145:907-23). However, the specifity of PSA assays for cancer detection is limited by the fact that levels above normal are found also in 30-50% of all patients with benign prostatic hyperplasia (BPH) (Stamey et al. New Engl J Med 1987; 317:909-16, Hudson et al. J Urol 1989; 142:1011-7). Serum concentrations of PSA inbetween 4-20 .mu.g/L are particularly common in BPH patients and in patients presenting with organ confined, potentially curable CAP. This reduces the utility of the PSA assay in early diagnosis of prostate cancer.
PSA is a serine protease of the glandular kallikrein family and occurs in serum predominantly in complex with the serine protease inhibitor alpha-1-antichymotrypsin (ACT) (Lilja et al. Clin Chem 1991; 37:1618-25). A small amount of PSA circulates in a free, non-complexed form despite a large excess of ACT in serum (Lilja et al. 1991). Most commercial PSA assays detect both free PSA and PSA-ACT complex which constitutes the total PSA fraction (PSA-T). However, all assays do not detect the two forms with equal potency (Stamey et al. J Urol 1994; 152:1-5). This might affect the ability to detect CAP as it has been shown that the proportion of PSA-ACT complex form is higher in serum of CAP patients compared to those with BPH (Stenman et al. Cancer Res 1991; 51:222-6, Christensson et al. J Urol 1993; 150:100-5). This is consistent with the detection of lower free-to-total PSA ratios in CAP than in BPH (Christensson et al. 1993). The reported data also show that specific measurements of the different PSA forms improve the early diagnosis of CAP.
Large amounts of PSA are produced by the glandular epithelium of the human prostate and secreted into the seminal fluid. Low concentrations of PSA are usually found in the general circulation. The serum concentration of PSA is increased in various disease states of the prostate, especially in CAP. PSA measured by immunoassays is the predominant marker in the monitoring of CAP, mainly due to high correlation with tumor volume. The American Cancer Society has recently recommended (Kramer et al. Ann Intern Med 1993; 119:914-23) that determinations of PSA combined with digital rectal examination should be applied to men over 50 years to improve the early detection of CAP. However, the diagnostic potential of PSA measurements in serum is limited by the fact that PSA levels also are elevated in many subjects with BPH. Several improvements have been suggested to enhance the diagnostic specificity of serum PSA determinations.
Experiments designed to detemine the sequence of 5' end of PSA mRNA revealed that the prostate expresses another highly similar gene, which was identified as hGK-1 (Chapdelaine et al. FEBS Lett 1988; 236:205-8). Both PSA and hGK-1 have gene structures shared by all tissue kallikreins. Their coding sequences are 85% similar, their introns 79-86% similar and their promoter regions 91% similar (Lundwall, Biochem Biophys Res Commun 1989; 161:1151-9, Riegman et al. Biochem Biophys Commun 1989; 159:95-102, Scheidlich et al. Clin Exp Pharmacol Physiol 1988; 15:339-44). The similarity between PSA and the predicted hGK-1 protein is very pronounced and higher than that between either PSA and KLK1 (kallikrein 1) or hGK-1 and KLK1. The prostatic expression of hGK-1 has been shown to be restricted to the secretory epithelial cells, and may be 10-50% lower than that of PSA as shown by the prostate levels of hGK-1 and PSA mRNA, respectively (Young et al. Biochemistry 1992; 31:818-24). So far, the prostate is the only identified location of hGK-1 gene expression. Thus, although the hGK-1 gene is expressed in the same prostatic epithelial cell as the PSA gene, the predicted hGK-1 protein remains to be characterized in vivo. However, the coding sequences of hGK-1 predict a 261 amino acid preprotein which would give rise to a mature glycoprotein of 237 residues.
PSA is a 33 kDa glycoprotein and the mature protein consists of 237 amino acids (Lundwall & Lilja, FEBA Lett 1987; 214:317-22, Riegman et al. Biochem Biophys Res Commun 1988; 155:181-8, Henttu et al. Biochem Biophys Res Commun 1989; 160:903-10). The amino acid sequence of mature PSA has also been confirmed by analysis of purified protein (Schaller et al. Eur J Biochem 1987; 170:111-20, Watt et al. Proc Natl Acad Sci USA 1986; 83:3166-70). The hGK-1 amino acid sequence predicts one asparagine-linked glycosylation site, but at a different position when compared to PSA. The hGK-1 protein remains to be isolated and characterized.
Certain structural features of PSA and the putative hGK-1 protein are shown in FIG. 1 (Henttu et al. Ann Med 1994; 26:165-71). It is likely that the three-dimensional structures of PSA and hGK-1 are very similar as both proteins retain the 10 cystein residues involved in the folding of kallikrein. There are six regions in PSA and hGK-1 polypeptide chains where 15 consecutive amino acids are identical. Therefore, it is very likely that PSA and hGK-1 share antigenic epitopes that are exposed in both proteins, as these regions also span hydrophilic regions in the proteins (Henttu et al. 1989).
In the present invention we have produced recombinant hGK-1 and PSA in vitro. The structures of the expressed proteins have been confirmed. Epitope maps for both proteins have been constructed employing a wide selection of monoclonal antibodies previously raised and characterized against PSA. Monoclonal antibodies were identified that share common antigenic epitopes on both PSA and hGK-1 in addition to monoclonal antibodies that recognize epitopes on PSA only. The identified monoclonal antibodies were used to design assays to measure total PSA+hGK-1 and total PSA alone. Serum samples from CAP and BPH patients were used to measure the concentrations of free PSA, total PSA, total PSA+hGK-1 and PSA-ACT. A clinical validation of the results was performed. Moreover, a separate assay was designed to measure only the hGK-1 concentration in serum samples. This is achieved by preincubating the sample with a PSA-specific monoclonal antibody to prevent PSA to be detected by an assay detecting both PSA and hGK-1.
Proteolytically active PSA slowly forms stable complexes in vitro with alpha-1-antichymotrypsin (ACT), alpha-2-macroglobulin, protein C inhibitor and pregnancy zone protein. In 1991 (Lilja et al. 1991) it was shown that PSA complexed to ACT is the major form of PSA in serum. An immunometric assay specific for the free non-complexed PSA was used to estimate that on average 15-20% of the total PSA concentration occurs in this form. Two-site immunometric assays of PSA-T (i.e. total PSA), PSA-ACT (i.e. PSA complexed to ACT) and PSA-F (i.e. free PSA) reported in this investigation were later used to demonstrate (Christensson et al. 1993), Lilja et al. International pat appl publ no WO 92/01936) that the ratios of PSA-F/PSA-T, being smaller in CAP than in BPH, or PSA-ACT/PSA-T, being greater in CAP than in BPH, improved the discrimination between the two groups of patients. Independent findings have later confirmed the original observation of the increased discrimination between CAP and BPH obtained by the measurement of the ratio of PSA-F/PSA-T. When, according to the present invention, the measurement of hGK-1 in addition to the different forms of PSA became possible, serum samples from CAP and BPH patients were analyzed in regard to the usefulness of hGK-1 or hGK-1 in combination with different forms of PSA in the discrimination of these two patient groups.